Neoplastic diseases, characterized by the proliferation of cells not subject to the normal control of cell growth, are a major cause of death in humans and other mammals. Clinical experience in cancer chemotherapy has demonstrated that new and more effective drugs are desirable to treat these diseases. Such clinical experience has also demonstrated that drugs which disrupt the microtubule system of the cytoskeleton can be effective in inhibiting the proliferation of neoplastic cells.
Cryptophycin compounds can now be prepared using a total synthetic process; see for example, Barrow, R. A. et al., J. Am. Chem. Soc. 117, 2479 (1995).
The processes claimed herein provide important elements needed for an efficient total synthetic route for preparing useful cryptophycin compounds and intermediates.
4-Hydroxy-5,6-dihydropyran-2-one and derivatives thereof are important intermediates for a number of natural products; D.Seebach et al., Angew. Chem. Int. Ed. 13, 77 (1974); R. M. Carlson et al., J. Org. Chem. 40, 1610 (1975). Additionally, this series of compounds has been used for the synthesis of pharmaceuticals, for example, the drug tetrahydrolipstatin (xe2x80x9cTHLxe2x80x9d); J. J. Landi, Jr. et al., Tetrahedron Lett., 34, 277 (1993). Current art teaches that in order to form a carbon-carbon bond at the terminal (4-) position of an acylacetate, two equivalents of strong base, for example sodium hydride and n-butyl lithium, in an aprotic solvent must be used to deprotonate both the 2- and 4-positions, proceeding through selective alkylation of a dianion with one equivalent of electrophiles; S. M. Huckin et al., Can. J. Chem. 52, 2157 (1974); S. M. Huckin et al., J. Am. Chem. Soc. 96, 1082 (1974); N. Petragnani et al., Synthesis, 521, 78 (1982); J. R. Peterson et al., Syn. Commun. 18, 949 (1988); D. Seebach et al., Angew. Chem. 86, 40 (1974); H. Kashihara et al., Chem. Pharm. Bull. 34, 4527 (1986).
However, even under such harsh conditions, paraformaldehyde or formaldehyde have been poor electrophiles and product yield has been low. In fact, a toxic reagent, PhCH2OCH2Cl has been used instead of paraformaldehyde for this purpose in a multistep synthesis; E. C. Taylor et al., J. Org. Chem. 50, 5223 (1985).
The present invention provides a process for making intermediates of cryptophycin compounds, including Fragment A analogs of cryptophycin, as well as a process for making cryptophycin compounds using selected intermediates.
The present invention further provides novel intermediates useful in the preparation of cryptophycin compounds.
The present invention provides a process for the preparation of a compound of the formula: 
wherein
G is C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ar;
Ar is an aromatic or heteroaromatic group or a substituted aromatic or heteroaromatic group;
R3 is C1-C6 alkyl;
R2a is trityl or a suitable silyl protecting group; and
Ra is hydrogen, allyl or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof;
said process comprising the steps of:
(a) contacting a compound of the formula: 
xe2x80x83wherein Rb is a suitable carboxy protecting group; and R3 is as defined above; with a cyclizing agent to form a compound of the formula: 
xe2x80x83wherein R3 is as defined above and M is hydrogen or a cation;
(b) stereoselectively reducing the compound of formula (3) with a stereoselective reducing agent to yield a compound of the formula: 
xe2x80x83wherein R3 is defined as above;
(c) reacting a compound of formula (4) with a hydroxy protecting agent to yield a compound of the formula: 
xe2x80x83wherein R2a is trityl or a suitable silyl protecting group, and R3 is as defined above;
(d) reacting the compound of formula (5) with a reducing agent followed by an olefinating reagent to form a compound of the formula: 
xe2x80x83wherein G, R3 and R2a are as defined above;
(e) reacting the compound of formula (6) with an oxidizing agent to provide a compound of the formula: 
xe2x80x83wherein G, R3 and R2a are as defined above; and
(f) reacting the compound of formula (7) with an alkyl ester forming agent, optionally with a hydrolyzing agent to provide a compound of formula (I) and optionally forming a pharmaceutically acceptable salt thereof.
This invention further comprises a process for preparing a cryptophycin compound using a compound of formula (I).
This invention further comprises the novel compounds of formulae (3), (4) and (5).
As used in the application:
(a) the designation  refers to a bond that protrudes forward out of the plane of the page;
(b) the designation  refers to a bond that protrudes backward out of the plane of the page; and
(c) the designation  refers to a bond for which the stereochemistry is not designated.
As used herein, the term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to either acid addition salts or base addition salts.
The expression xe2x80x9cpharmaceutically acceptable acid addition saltxe2x80x9d is intended to apply to any non-toxic organic or inorganic acid addition salt of the compounds of formula I or any of its intermediates. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric, and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate, and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include the mono-, di- and tricaboxylic acids. Illustrative of such acids are for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxy-benzoic, and sulfonic acids such as p-toluenesulfonic acid, methane sulfonic acid and 2-hydroxyethane sulfonic acid. Such salts can exist in either hydrated or substantially anhydrous form.
The expression xe2x80x9cpharmaceutically acceptable basic addition saltsxe2x80x9d is intended to apply to any non-toxic organic or inorganic basic addition salts of the compounds of formula I or any of its intermediates. Illustrative bases which form suitable salts include alkali metal or alkaline-earth metal hydroxides such as sodium, potassium, calcium, magnesium or barium hydroxides; ammonia and aliphatic, cyclic or aromatic organic amines such as methylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, isopropyldiethylamine, pyridine and picoline.
As used herein, the term xe2x80x9cC1-C12 alkylxe2x80x9d refers to a saturated straight or branched chain hydrocarbon group of from one to twelve carbon atoms. Included within the scope of this term are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, octyl, nonyl, decyl and the like. Included within the term is the term xe2x80x9cC1-C6 alkylxe2x80x9d which refers to a saturated, unsaturated, straight or branched chain hydrocarbon radical of from one to six carbon atoms. Included within the scope of this term are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, 2-methylbutyl, 3-methylbutyl, hexyl and the like. Included within the terms xe2x80x9cC1-C12 alkylxe2x80x9d and xe2x80x9cC1-C6 alkylxe2x80x9d is the terms xe2x80x9cC1-C3 alkylxe2x80x9d which refers to a saturated, unsaturated, straight or branched chain hydrocarbon radical of from one to three carbon atoms. Included within the scope of this term are methyl, ethyl, isopropyl, and the like.
xe2x80x9cSubstituted (C1-C6)alkylxe2x80x9d refers to a C1-C6 alkyl group that may include up to three (3) substituents containing one or more heteroatoms. Examples of such substituents are OH, NH2, CONH2, CO2H, PO3H2 and SO2R21 wherein R21 is hydrogen, C1-C3 alkyl or aryl.
The term xe2x80x9c(C3-C8)cycloalkylxe2x80x9d refers to a saturated C3-C8 cycloalkyl group. Included within this group are cyclopropyl, cyclobutyl, cyclohexyl, cyclooctyl, and the like. A xe2x80x9csubstituted (C3-C8)cycloalkyl groupxe2x80x9d refers to a (C3-C8)cycloalkyl group having up to three C1-C3 alkyl, halo, or OR21 substituents. The substituents may be attached at any available carbon atom. Cyclohexyl is an especially preferred cycloalkyl group. The term xe2x80x9cxe2x80x94(CH2)mxe2x80x94(C3-C5) cycloalkylxe2x80x9d where m is an integer one, two or three refers to a cyclopropyl, cyclobutyl or cyclopentyl ring attached to a methylidene, ethylidene or propylidene substituent.
The term xe2x80x9cC2-C12 alkenylxe2x80x9d refers to an unsaturated straight or branched chain hydrocarbon radical of from two to twelve carbon atoms and having from one to three triple bonds. Included within the scope of this term are ethenyl, propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, 2-methylbutenyl, 3-methylbutenyl, hexenyl, octenyl, nonenyl, decenyl and the like. It is especially preferred that alkenyl have only one double bond.
The term xe2x80x9cC2-C12 alkynylxe2x80x9d refers to an unsaturated straight or branched chain hydrocarbon radical of from two to twelve carbon atoms and having from one to three triple bonds. Included within the scope of this term are ethynyl, propynyl, isopropynyl, 2-methypropynyl, hexynyl, decynyl, and the like. It is particularly preferred that alkynyl has only one triple bond.
The term xe2x80x9cC1-C6 alkoxyxe2x80x9d refers to a straight or branched alkoxy group containing from one to six carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, pentoxy, 2-methylpentoxy, and the like. The term xe2x80x9c(C1-C6 alkoxy)phenylxe2x80x9d refers to a phenyl group substituted with a C1-C6 alkoxy group at any available carbon on the phenyl ring.
The term xe2x80x9chaloxe2x80x9d refers to chloro, bromo, fluoro, or iodo.
The terms xe2x80x9caromatic groupxe2x80x9d and xe2x80x9cheteroaromatic groupxe2x80x9d refer to common aromatic rings having 4n+2 pi electrons in a monocyclic or bicyclic conjugated system. The term xe2x80x9carylxe2x80x9d refers to an aromatic group, and the term xe2x80x9caralkylxe2x80x9d refers to an aryl(C1-C6-alkyl) group. Examples of aromatic groups are phenyl, benzyl and naphthyl. Heteroaromatic groups will contain one or more oxygen, nitrogen and/or sulfur atoms in the ring. Examples of heteroaromatic groups include furyl, pyrrolyl, thienyl, pyridyl and the like. When the aromatic or heteroaromatic groups are substituted, they may have from one to three independently selected C1-C6 alkyl, C1-C6-alkoxy or halo, substituents. The aromatic groups may be further substituted with trifluoromethyl, COOR57 (wherein R57 is hydrogen or C1-C6 alkyl) , PO3H, SO3H, SO2R57, N(R59)(R60) (wherein R59 is hydrogen or C1-C6 alkyl and R60 is hydrogen, C1-C6 alkyl, BOC or FMOC), xe2x80x94CN, xe2x80x94NO2, xe2x80x94OR57, xe2x80x94CH2OC(O) (CH2)mxe2x80x2NH2 (wherein mxe2x80x2 is an integer 1 to 6) or xe2x80x94CH2xe2x80x94Oxe2x80x94Si(R57)(R58) (R59) (wherein R58 is hydrogen or C1-C6 alkyl). Especially preferred substituents for the aromatic groups include methyl, halo, N(R59) (R60), and xe2x80x94OR57. The substituents may be attached at any available carbon atom.
Especially preferred heterocyclic or substituted heterocyclic groups include: 
wherein R20 is hydrogen or C1-C6 alkyl.
The term xe2x80x9cO-arylxe2x80x9d refers to an aryloxy or an aryl group bonded to an oxy moiety.
As used herein, the term xe2x80x9cTBSxe2x80x9d refers to tert-butyldimethylsilyl represented by the formula: 
As used herein, the term xe2x80x9cNHSxe2x80x9d refers to N-hydroxysuccinimide represented by the formula: 
As used herein the term xe2x80x9cPhxe2x80x9d refers to a phenyl moiety.
As used herein the term xe2x80x9cbase labile amino protecting groupxe2x80x9d refers to common amino protecting groups which are known to be base labile. The artisan can consult common works such as Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981). See particularly Chapter 7 of Greene. An especially preferred base labile amino protecting group is fluorenylmethoxycarbonyl (Fmoc).
The term xe2x80x9csuitable activatable carboxy protecting groupxe2x80x9d refers to carboxy protecting groups containing activatable ester substituents and are known by one of ordinary skill in the art and disclosed by Greene, T. W., supra. Suitable carboxy protecting groups are those which are activatable ester substituents including N-hydroxy-succinimide, N-hydroxysulfosuccinimide and salts thereof, 2-nitrophenyl, 4-nitrophenyl, 2,4-dichlorophenyl, and the like. An especially preferred activatable carboxy protecting group is N-hydroxy-succinimide (NHS).
As used herein, the term xe2x80x9ccryptophycin compoundxe2x80x9d refers to a compound of the formula: 
wherein
G and R3 are as defined in formula (I);
R1 is halogen and R2 is OH or glycinate ester; or R1 and R2 may be taken together to form an epoxide ring; or R1 and R2 may be taken together to form a bond;
R7 and R8 are each independently hydrogen or C1-C6 alkyl; or
R7 and R8 taken together form a cyclopropyl or cyclobutyl ring;
R9 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, xe2x80x94(CH2)mxe2x80x94(C3-C5)cycloalkyl or benzyl, wherein m is the integer one to three;
R10 is hydrogen or C1-C6 alkyl;
R11 is hydrogen, C1-C6 alkyl, phenyl or benzyl;
R14 is hydrogen or C1-C6 alkyl;
R50 is hydrogen or (xe2x95x90O);
Y is CH, O NR12, S, SO, SO2, wherein R12 is H or C1-C3 alkyl;
R6 is C1-C6 alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, a heteroaromatic or substituted heteroaromatic group or a group of formula (IA), (IB) or (IC): 
R6a, R6b, and R6c independently are H, (C1-C6)alkyl, halo NR18R19 or OR18;
R15, R16, and R17 independently are hydrogen, halo, (C1-C6)alkyl, OR18, O-aryl, NH2, NR18R19, NO2, OPO4H2, (C1-C6 alkoxy)phenyl, S-benzyl, CONH2, CO2H, PO3H2, SO2R23, or Zxe2x80x2;
R18 and R19 independently are hydrogen or C1-C6 alkyl;
R23 is hydrogen or (C1-C3)alkyl;
Z is xe2x80x94(CH2)nxe2x80x94 or (C3-C5)cycloalkyl;
n is 0, 1, or 2; and
Zxe2x80x2 is an aromatic or substituted aromatic group; or a pharmaceutically acceptable salt thereof.
As used herein, the term xe2x80x9cCryptophycin 52xe2x80x9d represents the compound of the formula: 
A general synthetic procedure for preparing a compound of formula (I) is set forth in Scheme A. In Scheme A, all substituents unless otherwise indicated, are as previously defined. Reagents, techniques, and procedures used in Scheme A are well known and appreciated by one of ordinary skill in the art. 
In Scheme A, step 1, the acylacetate of formula (2) is cyclized with a suitable cyclizing agent to form a lactone of formula (3).
A suitable cyclizing agent is any agent capable of converting the acylacetate of formula (2) to the lactone of formula (3).
For example, an acylacetate of formula (2) is added to a solution of a suitable base, such as potassium t-butoxide, lithium dialkylamides, for example, lithium diisopropylamide, sodium hydride and the like. Most preferred is potassium t-butoxide. The suitable base is dissolved in suitable organic solvent, for example, alcoholic solvents, such as methanol, ethanol, 2-propanol, or mixtures thereof; tetrahydrofuran, and the like. Most preferred are alcoholic solvents, such as 2-propanol. The amount of suitable base to be dissolved ranges from about 1.0 molar equivalents to about 2.0 molar equivalents as compared to the acylacetate of formula (2). Preferably, the amount of suitable base ranges from about 1.3 to about 1.7 molar equivalents. Most preferably, the amount of suitable base ranges from about 1.4 to about 1.6 molar equivalents. The basic solution is set to a temperature ranging from about xe2x88x9230xc2x0 C. to about 30xc2x0 C., preferably under an inert atmosphere, such as nitrogen, in preparation for the reaction with the desired acylacetate of formula (2). Most preferably, the solution is cooled to about 0xc2x0 C.
The acylacetate of formula (2) is added to the basic solution at a rate so as to maintain the temperature at or below +10xc2x0 C. Preferably, the acylacetate of formula (II) is added so as to maintain the temperature between xe2x88x925xc2x0 C. and +7xc2x0 C. Most preferably, the acylacetate of formula (II) is added so as to maintain the temperature between 0xc2x0 C. and +5xc2x0 C.
The acylacetate basic solution is then reacted with a suitable aldehyde or ketone of formula (2bxe2x80x2), which corresponds to the compound of formula (2b) wherein R2b is hydrogen. The amount of aldehyde or ketone of formula (2bxe2x80x2) to be added ranges from about 1.0 molar equivalents to about 3.0 molar equivalents as compared to the acylacetate of formula (2). Preferably, the amount of suitable base ranges from about 1.1 to about 2.2 molar equivalents. Most preferably, the amount of suitable base ranges from about 1.2 to about 1.5 molar equivalents. Generally, the aldehyde or ketone of formula (2bxe2x80x2) is reacted with the acylacetate solution at a temperature ranging from about 0xc2x0 C. to about 50xc2x0 C. Most preferably, the reaction is carried out at room temperature.
The resulting mixture is then acidified with a suitable acid, such as hydrochloric acid. The acidified mixture is then isolated and purified according to methods appreciated by one of ordinary skill in the art, such as extraction, evaporation, filtration and recrystallization to provide the lactone of formula (3).
The acylacetates of formula (2) are known or readily prepared by one of ordinary skill in the art. Examples include ethyl 2-methylacetoacetate, ethyl 2-n-hexylacetoacetate, ethyl 2-ethylacetoacetate, ethyl 2-n-propylacetoacetate, ethyl 2-isopropylacetoacetate, and the like.
The preferred aldehydes or ketones of formula (2bxe2x80x2) include paraformaldehyde, acetaldehyde, acetone, and the like.
In Scheme A, step 2, the lactone of formula (3) is contacted with a stereoselective reducing agent to provide the stereoselectively reduced compound of formula (4).
The stereoselective reducing agent used in Scheme A, step 2 may be either chemical, or preferably biological. In the case of biological agents, the preferred agents are microorganisms which contain reducing enzymes, more preferred microorganisms of genus Mortierella. Particular preference is given to the species: Mortierella isabellina, Mortierella alpina, Mortierella pusilla, Mortierella nana, Mortierella vinacea, and Mortierella ovata. Most preferably, the microorganism is Mortierella isabellina ATCC 42613. Other suitable biological agents for this process include the genera: Pichia, Saccharomyces, Candida, Kluyveromyces, Zygosaccharomyces, Pichia, Aureobasidium, Torulopsis, Trigonopsis, Kloeckeva, Hanseniaspora, Schizosaccharomyces, Cryptococcus, Rhodotorula, Geotrichum, Rhizopus and Cumminghamella. Selected species of these genera were tested for the preparation of formula (4) and did not provide significant yield under the conditions tried: Zygosaccharomyces rouxii ATCC 14462, Candida guillermondi ATCC 2479, Pichia fermentens ATCC 10651, Nematospora coryli NRRL Y-1343, Candida famata ATCC 26418, Saccharomyces pastorianus ATCC 2366 , Saccharomyces uvarum ATCC e9080, Candida utilis ATCC 9950, Saccharomyces globosus ATCC 10600, Kluyveromyces dobzhanskii NRRL-Y-1974, Kluyveromyces lactis QM 8230, Aureobasidium pullulans QM 2725, Kloeckeva javanica ATCC 10636, Hanseniaspora valbyensis ATCC 10631, Octosporomyces octosporus ATCC 10631, Candida parapsilosis ATCC 22019, Candida tropicalis ATCC 12659, Torulopsis taboadae ATCC 42213, Torulopsis ethanolitolerans ATCC 46859, Torulopsis ethanolitolerans ATCC 46859, Torulopsis ptarmiganii ATCC 26902, Torulopsis sonorensis ATCC 56511, Trigonopsis variabilis ATCC 10679, Torulopsis enokii ATCC 20432, Candida boidinii ATCC 18810, Candida blankii ATCC 18735, Cryptococcus laurentii ATCC 42922, Hansenula polymorpha ATCC 34438, Rhodotorula mucilaginosa A35210, Kluyveromyces marxianus ATCC 8554, Saccharomyces bayanus ATCC 76516, Sporobolomyces salmonicolor ATCC 26697, Cryptococcus laurentii ATCC 36833, Arthroascus javanensis NRRL Y1493, Hyphopicia burtonii NRRL Y1988, Saccharomycopsis capularis NRRL Y50, Yarrowia lipolytica NRRL YB423-3, Guillermondella selenospora NRRL Y1357, Saccharomycopsis fibuligera NRRL Y3, Lipomyces tetrasporus NRRL 7074, Pachysolen tannophilus NRRL 2460, Geotrichum candidum ATCC 7471 or ATCC 14253, Ambrosiozyma monospora ATCC 14628, Chinosphaera apobasidialis ATCC 52639, Phaffia rhodozyma ATCC 24202, Debaryomyces polymorphus ATCC 20499, Endomycopsella vini ATCC 34382, Schizosaccharomyces pombe ATCC 26189, Schwanniomyces occindentalis ATCC 26077, Bensingtonia yuccicola ATCC 66429, Rhizopus oryzae ATCC 9363, Rhizopus stolonifer A33417, Mortierella ramanniana ATCC 38191, Mortierella verticillata NRRL 6337, Mortierella chlamydospora NRRL 2769, Mortierella multidivaricata ATCC 58767, Mortierella sepedonioides NRRL 6425, Mortierella elongata NRRL 5513, Mortierella sp. NRRL 1458, Mortierella hyalina NRRL 6427, Mortierella pulchella ATCC 18078, Mortierella bisporalis NRRL 2493, Mortierella sclerotiella ATCC 18732, Mortierella minutissima ATCC 16268, Mortierella spinosa ATCC 16272 Penicillium glabrum ATCC 11080, Emericella quadrilineata ATCC 12067, Syncephalastrum racemosum ATCC 20471, Geotrichum sp. ATCC 32345, Aspergillus niveus ATCC 20922, Aspergillus niger ATCC 64958, Cunninghamella echinulata var.echinulata ATCC 36190, Mucor circinelloides f.circinelloides ATCC 15242, Penicillum purpurogenum ATCC 9777, Beauveria bassiana ATCC 9835, Nocardia salmonicolor ATCC 19149, Rhizopus nigricaus ATCC 6227b, Mortierella epigama ATCC 2402, and Mortierella schmuckeri ATCC 42658.
Zygosaccharomyces rouxii ATCC 14462 and Candida guillermondi ATCC 2479, gave indication on GC that they produced the hydroxylactone from the ketone. However, significant ketone remained indicating that conversion was poor compared to Mortierella under the conditions tested.
For example, a suitable microorganism, such as Mortierella isabellina ATCC 42613 may be used in free state as wet cells, freeze-dried cells or heat-dried cells. Immobilized cells on support by physical adsorption or entrapment can also be used. Appropriate media for growing microorganisms for this process typically include necessary carbon sources, nitrogen sources, and trace elements. Inducers may also be added. As used herein, the term xe2x80x9cinducerxe2x80x9d refers to any compounds having keto or aldehyde groups, such as paraformaldehyde and the like.
Carbon sources include sugars such as maltose, lactose, dextrose, glucose, fructose, glycerol, sorbitol, sucrose, starch, mannitol, propylene glycol, and the like; organic acids such as sodium acetate, sodium citrate, and the like; amino acids such as sodium glutamate and the like; alcohols such as ethanol, propanol, and the like.
Nitrogen sources include N-Z amine A, corn steep liquor, soy bean meal, beef extracts, yeast extracts, molasses, baker""s yeast, tryptone, nutrisoy, peptone, yeastamine, sodium nitrate, ammoonium sulfate, and the like.
Trace elements include phosphates, magnesium, manganese, calcium, cobalt, nickel, iron, sodium, and potassium salts.
For the purposes of this invention, appropriate media may include more than one carbon or nitrogen source and may include a mixture of several.
After sterilization the pH of the medium should be adjusted to 4.5 to 6.5, preferably 5.5. The pH may be maintained between about 4.0 and 6.0, preferably at 5.5 during the fermentation and 4.5 during the bioreduction.
The temperature of the reaction mixture should be maintained to ensure that there is sufficient energy available for the process. The temperature is a measure of the heat energy available for the transformation process. A suitable temperature of reaction ranges from about 20xc2x0 C. to 35xc2x0 C. A preferred temperature range is from about 25xc2x0 C. to about 30xc2x0 C.
The agitation and aeration of the reaction mixture affects the amount of oxygen available during the fermentation and bioreduction stages of the process. During both stages the agitation range from 150 to 450 rpm is preferable, with 150 to 275 rpm being most preferred. Aeration of about 0.5 to 3.5 standard cubic feet per minute (scfm) is preferable, with 0.5 to 1.0 scfm being most preferred.
The reaction time for the reduction of Scheme A, step 2 ranges from about 24 to 96 hours, preferably 24 to 48 hours, measured from the time of initially treating the substrate (3) with the microorganism to provide the lactone of formula (4).
In Scheme A, step 3, the lactone of formula (4) is reacted with a hydroxy protecting agent to yield the protected lactone of formula (5).
A suitable hydroxy protecting agent includes compounds of the formula R2a-LG where R2a is trityl or a silyl protecting group, preferably tri(C1-C6 alkyl)silyl, and LG is a suitable leaving group, such as a halogen or a sulfonate, such as trifluoromethanesulfonate. Specific examples of hydroxy protecting agents include t-butyldimethylsilyl chloride, t-butyldimethylsilyl trifluoromethane sulfonate, chlorotrimethylsilane and the like.
For example, the lactone of formula (4) is contacted with a suitable base, most preferably imidazole, in a suitable organic solvent such as CH3CN. A suitable hydroxy protecting agent, such as t-butyldimethylsilyl chloride, is then added to the solution, optionally with a suitable coupling catalyst such as dimethylaminopyridine. The mixture is then stirred at a temperature of from about 0xc2x0 C. to about 60xc2x0 C., preferably room temperature, for a period of time ranging from about 2 to 24 hours. The protected alcohol of formula (5) can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation. The product can be purified by chromatography and recrystallization.
In Scheme A, step 4, the protected alcohol of formula (5) is reacted with a reducing agent followed by an olefinating agent to provide the olefin of formula (6).
A suitable reducing agent includes alkylated aluminum hydrides and other reagents that would convert the protected lactone of formula (5) into a lactol and/or open chained hydroxyaldehyde intermediate. Examples include diisobutylaluminum hydride, bis(dialkylamino)aluminum hydride, either preformed or generated in situ from alkali-aluminum compounds such as LiAlH4, NaAlH4, NaH2Al(C1-C6 alkyl)2, NaH2Al (OCH2CH2OMe)2 LiHAl(OtBu)2 and the like, in combination with dialkyl or cyclic amines such as dimethylamine, diethylamine, dipropylamine, morpholine, piperidine and the like.
A suitable olefinating agent includes aryl Wittig reagents, aryl Horner-Emmons Wadsworth reagents and other reagents that are known by one of ordinary skill in the art to convert aldehydes to olefins in either a one-step or stepwise fashion. Examples include benzyldiphenylphosphine oxide (BDPPO), triphenyl benzyl phosphonium chloride and the like. The synthesis of suitable olefinating agents are known in the art. For example, the synthesis of BDPPO is described by Brown, Tetrahedron Lett. 35 (36), 6733 (1994).
For example, the protected lactone of formula (5) is reacted with a suitable reducing agent such as DIBAL or DIBAH under an inert atmosphere, for a period ranging from about 0.5 to 12 hours. The reaction is carried out in the presence of a suitable organic solvent, such as methylene chloride or hexane while the temperature is maintained below xe2x88x9210xc2x0 C. to form portion A. In a separate reaction vessel a suitable olefinating agent, such as BDPPO or triphenyl benzyl phosphonium chloride is contacted with a suitable base, such as sodium bis(trimethylsilyl)amide or potassium tert-butoxide in the presence of a suitable organic solvent such as tetrahydrofuran (THF) or methylene chloride. The solution may be stirred at room temperature for a period of time ranging from about 10 minutes to 2 hours. The resulting reddish solution is then contacted with portion A and stirred for 1 to 36 hours at a temperature ranging from about 0xc2x0 C. to about 70xc2x0 C. The olefin of formula (6) may be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation. The product can be purified by chromatography and recrystallization.
In Scheme A, step 5, the olefin of formula (6) is oxidized with an oxidizing agent to provide the aldehyde of formula (7).
An oxidizing agent is a reagent capable of converting the hydroxy moiety on the olefin of formula (6) to aldehyde moiety of formula (7). Suitable oxidizing agents include oxalyl chloride/DMSO, TEMPO/NaOCl, P2O5/DMSO, (COCl)2/DMSO, NBS/TEMPO, and the like.
For example, anhydrous dimethylsulfoxide (DMSO) is added to oxalyl chloride in a suitable organic solvent, such as methylene chloride over a period of time ranging from about 1 to about 30 minutes at a temperature ranging from about xe2x88x9230xc2x0 C. to about xe2x88x9278xc2x0 C., preferably about xe2x88x9260xc2x0 C. The mixture is then stirred for a period of time ranging from about 10 minutes to 2 hours after which a solution of olefin for formula (6) in a suitable organic solvent, such as methylene chloride is added. After additional stirring for a period ranging from 5 to 30 minutes, a suitable base, such as triethylamine is added and the reaction is allowed to warm to room temperature. The aldehyde of formula (7) may be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation. The product can be purified by chromatography and recrystallization.
In Scheme A, step 6, the aldehyde of formula (7) is reacted with an alkyl ester forming agent to form the ester of formula (ID).
An alkyl ester forming agent is any agent capable of converting the aldehyde moiety of the compound formula (7) to the alkyl ester moiety of the compound of formula (ID), while inert to the other substituents on the molecules. For example, the aldehyde of formula (7) may be converted to the ester of formula (ID) by means of a Horner-Emmons reaction. Suitable examples of alkyl ester forming agents include trimethyl phosphonoacetate, (CH3O)2POCH2CH3 and the like.
For example, the aldehyde of formula (7) is contacted with an alkyl ester forming agent, such as trimethylphosphonoacetate, and tetramethylguanidine in a suitable organic solvent, such as tetrahydrofuran at ambient temperature and stirred for a period ranging from about 1 to about 24 hours. The ester of formula (ID) may be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation. The product can be purified by techniques well known in the art, such as chromatography.
In Scheme A, step 7, the ester of formula (ID) is reacted with a hydrolyzing agent to provide the acid of formula (IE).
A hydrolyzing agent is any agent that is capable of converting the ester moiety of the compound of formula (IA) to the acid moiety of the compound of formula (IB), while inert to the other substituents on the molecules. Examples of suitable hydrolyzing agents include inorganic bases such as sodium hydroxide and potassium hydroxide, with potassium hydroxide being preferred.
For example, the ester of formula (IA) is contacted with a suitable hydrolyzing agent, such as 2N KOH in a suitable organic solvent, such as 1,4-dioxane at ambient temperature. The solution is then heated to reflux for a period of time ranging from about 1 to about 6 hours. The reaction is then quenched with a suitable acid, such as 2N HCl. The acid of formula (IB) is isolated by techniques well known in the art, such as extraction, evaporation, and precipitation. The product can be purified by techniques well known in the art, such as chromatography.
General synthetic procedures for preparing cryptophycin compounds of formula (II) are set forth in Barrow, R. A. et al., J. Am. Chem. Soc. 117, 2479 (1995); PCT Intnl. Publ. No. WO 96/40184, published Dec. 19, 1996; PCT Intnl. Publ. No. WO 98/08505, published Mar. 5, 1998; PCT Intnl. Publ. No. WO 97/07798, published Mar. 6, 1998; PCT Intnl. Publ. No. WO 97/23211, published Jul. 3, 1997; PCT Intnl. Publ. No. WO 98/08506, published Mar. 5, 1998; PCT Intnl. Publ. No. WO 98/08812, published Mar. 5, 1998; and PCT Intnl. Publ. No. WO 97/31632, published Sep. 4, 1997. References disclosing intermediates and/or processes for preparing cryptophycin compounds of formula (II) or intermediates thereof include PCT Intnl. Publ. No. WO 98/09955, published Mar. 12, 1998; PCT Intnl. Publ. No. WO 98/09974, published Mar. 12, 1998; PCT Intnl. Publ. No. WO 98/09601, published Mar. 12, 1998; and PCT Intnl. Publ. No. WO 98/09988, published Mar. 12, 1998.
Scheme B illustrates a general synthetic procedure for preparing a cryptophycin compound of formula (II). In Scheme B, all substituents unless otherwise indicated, are as previously defined. As used herein, Rp is hydrogen or a suitable activatable carboxy protecting group; Rp1 is hydrogen or C1-C6 alkyl; R81 is C1-C6 alkyl, C3-C8 cycloalkyl, phenyl or benzyl; R82 is a base labile protecting group; Hal is halogen, preferably chloro, bromo or iodo; and q is an integer 1 or 2. 
In Scheme B, step 1, a compound of formula (IE) is optionally treated with a carboxy activating agent to provide the activatable ester of formula (8).
For example, a compound of formula (IE) is reacted with a suitable coupling agent, such as a carbodiimide, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and a suitable carboxy activating agent, such as N-hydroxysuccinimide, in a suitable organic solvent, such as dry dimethylformamide. The mixture is stirred for a period of time ranging from about 6 to 36 hours at a temperature ranging from about 10xc2x0 C. to about 50xc2x0 C. The activatable ester of formula (8) is isolated by techniques well known in the art, such as extraction, evaporation, and precipitation. The product can be purified by techniques well known in the art, such as chromatography.
In Scheme B, step 2, an activatable ester of formula (8) is epoxidized with an epoxidizing agent to form an epoxide of formula (9).
The compound of activatable ester of formula (8) may be epoxidized non-selectively using a suitable epoxidizing agent. An xe2x80x9cepoxidizing agentxe2x80x9d is an agent capable of converting the activatable ester of formula (8) into the epoxide of compound (9). Suitable epoxidizing agents include potassium peroxomonosulfate (oxone) in combination with acetone, m-CPBA, methyltrioxorhenium(VII), trifluoroper-acetic acid, and magnesium monoperoxyphthalate, with Oxone in combination with acetone, or m-CPBA being preferred. Possible solvents for the epoxidation activatable ester of formula (8) include acetone, DMF, glyme, dioxane, CH3CN, alcohols, THF, EtOAc, halohydrocarbons, chlorobenzene, dichloromethane and toluene. The reaction optionally takes place in the presence of a suitable base such as NaHCO3. Reaction temperatures may range from about xe2x88x9230xc2x0 C. to about 50xc2x0 C. with about xe2x88x9210xc2x0 C. to about 25xc2x0 C. being preferred. The xcex2-epoxide of formula (9) may be isolated and purified according to techniques and procedures well known in the art such as column chromatography. Either the xcex1- and xcex2-epoxides of formula (9) may be further separated by HPLC. It is preferred that the xcex2-epoxide of formula (9), is separated from the xcex1-epoxide of formula (9a), and further used in the remaining steps of the process of this invention to form a the xcex2-epoxy form of a compound of formula (I). However, the epoxidizing reaction of Scheme B, step 1 can also be used with the xcex1-epoxide of formula (9a) or with a mixture of the two epoxides. 
The compound of formula (I) wherein Ra is H may be epoxidized directly using m-CPBA. The m-CPBA epoxidation may be carried out on a compound of formula (I) to give a 1.2:1 b/a diastereomeric mixture of epoxides. The individual xcex1-and xcex2-diastereomers may be separated by HPLC, as described above. This direct epoxidation is illustrated in Scheme B1. 
By eliminating the use of the N-hydroxysuccinimide ester, one step is eliminated from the synthesis.
Furthermore, a compound of formula (9e) may be prepared by deesterifying a compound (9d) according to Scheme B2. In Scheme B2, Ra is C1-C6 alkyl whereas all of the remaining substituents are as previously defined. 
In Scheme B2, the alkyl ester of formula (9d) is deesterified with a suitable deesterifying agent to form the acid of formula (9e). The term xe2x80x9csuitable deesterifying agentxe2x80x9d encompasses any suitable means or conditions for removing the ester moiety of Ra while inert to the epoxide. For example, a suitable base, such as potassium hydroxide, is added to a solution of the alkyl ester of formula (9d) in a suitable solvent, such as tetrahydrofuran. The biphasic mixture is then allowed to stir at a temperature ranging from about 20xc2x0 C. to about 80xc2x0 C., preferably 40xc2x0 C. and 65xc2x0 C., for a period of from about 6 to 24 hours. After cooling to room temperature, the aqueous layer is washed with an appropriate acid, such as 1N hydrochloric acid, followed by brine. The mixture is dried, filtered and concentrated to provide the acid of (9e).
A compound of formula (I) or formula (8) may also be stereoselectively epoxidized to form either the compound of formula (9) or (9a) using a chiral ketone with Oxone in the presence of a suitable base such as NaHCO3 based on procedures analogous to those disclosed by Tu, Y. et al, J. Am. Chem. Soc. 118, 9806 (1996); Wang, Z-X et al. J. Org. Chem. 62, 2328 (1997); Wang, Z-X et al., J. Am. Chem. Soc. 119, 11224 (1997). Preferred compounds of formula (8) for this reaction include those compounds where G is phenyl, R3 is methyl, and R is NHS (N-hydroxysuccinimide). As used herein, the term xe2x80x9cchiral ketonexe2x80x9d refers to a ketone containing the following general features:
1) the stereogenic centers are close to the reacting center; and
2) the ketone has a fused ring and a quaternary center adjacent to a carbonyl group; and
3) one face of the ketone is sterically blocked.
One especially preferred chiral ketone is of the structure: 
This preferred chiral ketone can be prepared from D-fructose by ketalization and oxidation under routine conditions. For example, the ketalization can be completed using acetone, HClO4, and the process is conducted at about 0xc2x0 C. For example, the oxidation can be completed using pyridinium chlorochromate at room temperature. These reactions are known in the art; see, for example: Tu, Y. et al, supra. and Wang, Z-X et al. supra. The asymmetric epoxidation can be carried out at a pH within the range of from about 7.0 to about 11.5 during the reaction.
Although it requires about 3-4 equivalents of chiral ketone to obtain conversions of greater than 95% with many cryptophycin intermediates at a pH of about 8.0, it is possible to use less chiral ketone (about 1-2 equivalents) at a pH of about 9.0 or above. Suitable solvents useful for the epoxidation step include H2O, DMF, glyme, dioxane, CH3CN, alcohols, THF, EtOAc, halohydrocarbons, chloro-benzene, and toluene, with a CH3CN/H2O solvent combination being preferred. Reaction temperatures may range from about xe2x88x9220xc2x0 C. to about 25xc2x0 C. with about xe2x88x9210xc2x0 C. to about 10xc2x0 C. being preferred. The individual isomers, (9) or (9a), can be isolated from the crude mixture of isomers and purified by techniques well known in the art such as extraction, evaporation, chromatography and recrystallization. A preferred stereoselective epoxidation utilizes the chiral ketone of structure (9f) to provide a mixture of epoxides in the crude product in the ratio of about xcex1:xcex2 1:5.
The xcex2-epoxide of formula (9) is generally preferred and is used throughout the process of this invention.
In Scheme B, step 3, the epoxide of formula (9) is coupled to the amino acid of formula: 
wherein R6 and R14 are as defined above and Rp1 is hydrogen or C1-C6 alkyl to yield a Fragment A-B compound of formula (10).
The amino acids of formula (9g) are commercially available or are readily prepared by methods known in the art. Particularly preferred amino acids of formula (9g) include those where R6 is a group of formula (IA) and R6a is methoxy, R6b is chloro and R6c is H; R14 is hydrogen; and Rp1 is hydrogen; said amino acids being disclosed by PCT Intnl. Publ. No. WO 97/07798, published Mar. 6, 1997, PCT Intnl. Publ. No. WO 96/40184, published Dec. 19, 1996; Barrow, R.A. et al. J. Am. Chem. Soc. 117, 2479 (1995).
The epoxide of formula (9), where Rp is NHS, is coupled to the amino acid of formula (9g) according to coupling procedures which are inert to the epoxide functionality. For example, the epoxide of formula (9) is contacted with from about 1.5 to 3.5 equivalents of amino acid (9g), where Rp1 and R14 are both hydrogen, and a suitable silylating agent in the presence of a suitable organic solvent. Suitable organic solvents include DMF, glyme, dioxane, CH3CN, THF, EtOAc, and halohydrocarbons, such as methylene chloride. The reaction is carried out at a temperature ranging from about xe2x88x9230xc2x0 C. to about 75xc2x0 C., with a temperature ranging from about 20xc2x0 C. to about 60xc2x0 C. being preferred. The fragment A-B compound of formula (10) may be isolated and purified according to techniques and procedures well known in the art such as extraction, evaporation, chromatography and recrystallization.
As used herein, the term xe2x80x9csilylating agentxe2x80x9d is selected from any reagent capable of attaching a silyl group to a target substituent. Generally known silylating agents are employed. See for example, Calvin, E. W., xe2x80x9cSilicon Reagents in Organic Synthesisxe2x80x9d, Academic Press, (London, 1988). Generally typical silyl agents include any reagent with a trialkylsilyl group such as trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, and t-butyldimethylsilyl, any reagent with an alkylarylsilyl group such as tribenzylsilyl, diphenylmethylsilyl, t-butylmethoxyphenylsilyl and tri-p-xylylsilyl, and any reagent with a triarylsilyl group such as triphenylsilyl. The preferred silylating agent is a trimethyl silylating agent. Typical trimethyl silylating agents include N,O-Bis(trimethyl silyl) acetamide, allyltrimethylsilane, N,O-Bis(trimethylsilyl)-carbamate N,N-Bis(trimethylsilyl)methylamine, Bis(trimethylsilyl)sulfate, N,O-Bis(trimethylsilyl)trifluoroacetamide, N,N-Bis(trimethylsilyl)urea, (ethylthio)trimethylsilane, ethyl trimethyl silylacetate, hexamethyldisilane, hexamethyldisilazane, hexamethyldisiloxane, hexamethyldisilthiane, (isopropenyloxy)trimethyl silane, 1-methoxy-2-methyl-1-trimethyl-siloxy-propene, (methylthio)trimethylsilane, methyl 3-trimethylsiloxy-2-butenoate, N-methyl-N-trimethylsilylacetamide, methyl trimethylsilylacetate, N-methyl-N-trimethylsilyl-hepta-fluorobutyramide, N-methyl-N-trimethylsilyl-trifluoroacetamide, (phenylthio)trimethylsilane, trimethylbromosilane, trimethylchlorosilane, trimethyliodosilane, 4-trimethylsiloxy-3-penten-2-one, N-(trimethylsilyl)acetamide, trimethylsilyl acetate, trimethylsilyl azide, trimethylsilyl benzenesulfonate, trimethylsilyl cyanide, N-trimethylsilyldiethylamine, N-trimethylsilyldimethylamine, trimethylsilyl N,N-dimethylcarbamate, 1-(trimethylsilyl)imidazole, trimethylsilyl methanesulfonate, 4-(trimethylsilyl)morpholine, 3-trimethylsilyl-2-oxazolidinone, trimethylsilyl trichloroacetate, trimethylsilyl trifluoroacetate and trimethylsilyl trifluoromethane sulfonate. Particularly useful silylating agents include xe2x80x9ctri-lower alkyl silylxe2x80x9d agents, the term of which contemplates triisopropylsilyl, trimethylsilyl and triethylsilyl, trimethylsilyl halides, silylated ureas such as bis(trimethylsilyl)urea (BSU) and silylated amides such as N,O-bis(trimethylsilyl)acetamide (BSA). Bis N,O-trimethyl silyl acetamide (BSA) is an especially preferred silylating agent.
Alternatively, the desired xcex2-epoxide (9c) may be coupled with (9g), when Rp1 is hydrogen, using a suitable coupling agent, preferably diphenylphosphinic chloride, and a silyl agent to give fragment A-B (10). Suitable coupling agents are well known in the art, as described by Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981) and include N,O- diphenylphosphinic chloride, diphenyl chlorophosphate, DCC, EDCI, chloroformates, and 2-chloro-4,6-dimethoxy-1,3,5-triazine. Diphenylphosphinic chloride is a preferred coupling agent. The suitable organic solvents described above, preferably methylene chloride, may be used. This procedure allows for the elimination of the carboxy protection step and allows for the use of lower amounts of amino acid (9g).
In Scheme B, step 4, the fragment A-B compound of formula (10) is deprotected with a suitable alkoxy deprotecting agent to form a compound of formula (11).
A suitable alkoxy deprotecting agent is one that removes the hydroxy protecting group signified by the R2a substituent while inert to the epoxide moiety of the fragment A-B compound of formula (10). Preferred deprotecting agents include basic fluoride sources such as tetrabutylammonium fluoride, pyridinium fluoride, triethylammonium fluoride, cesium fluoride, and the like, with tetrabutylammonium fluoride being preferred. The deprotection reaction takes place in the presence of a suitable organic solvent such as tetrahydrofuran, optionally in the presence of a suitable base, such as sodium bicarbonate (NaHCO3). The reaction takes place in the range of from about 0xc2x0 C. to about 80xc2x0 C. with from about 20xc2x0 C. to about 70xc2x0 C. being preferred. The reaction is run for a period of time ranging from about 3 to 24 hours. Crude product (11) may be used without further purification. Alternatively, the compound of formula (11) may be isolated and purified according to procedures well known well known in the art such as extraction, evaporation, chromatography and recrystallization.
When Rp1 for the compound of formula (11) is hydrogen, the Rp1 moiety is actually the cationic salt of deprotecting agent, for example, cesium, tetrabutylammonium, and the like.
In Scheme B, step 5, the compound of formula (11) is contacted with a thioester forming agent to provide the ester of formula (12).
The term xe2x80x9cthioester forming agentxe2x80x9d encompasses any suitable means or conditions for forming the thioester moiety of formula (12). Included within this definition are the conditions set forth and/or analogously described in Ono, N. et al., Bull. Chem. Soc. Jpn. 51 (8), 2401 (1978); Ho, Tse-Lok, Synth. Comm. 9(4), 267-270 (1979); Narasaka, K. et al., J. Am. Chem. Soc. 106 (10), 2954-2960 (1984); L. G. Wade, Jr. et al., Tetrahedron Lett. 731-732 (1978); Mora, N. et al., Tetrahedron Lett. 34 (15), 2461-2464 (1993); and Dossena, A. et al. J. Chem. Soc. Perkin Trans. I, 2737 (1981).
For example, the compound of formula (11) may be treated with a sterically hindered alkyl halide, such as tert-butylbromide, and a solvent of the formula (R81) (Me) SO, wherein R81 is C1-C6 alkyl, C3-C8 cycloalkyl, phenyl or benzyl, in the presence of a suitable base, such as sodium bicarbonate (NaHCO3). A preferred solvent for reaction is dimethylsulfoxide (DMSO). Both the sterically hindered alkyl halide and the suitable base are added in a molar excess of about 7.0 to 12.0 in comparison to the compound of formula (11). The reaction takes place in the range of from about 0xc2x0 C. to about 60xc2x0 C. with from about 10xc2x0 C. to about 30xc2x0 C. being preferred. The reaction is run for a period of time ranging from about 1 to 24 hours. Crude product (12) may be used without further purification. Alternatively, the ester of formula (12) may be isolated and purified according to procedures well known in the art such as extraction, evaporation, chromatography and recrystallization.
In those instances when the substituent Rp1 is a moiety other than hydrogen, the compound of formula (11) must first be carboxy-deprotected. Carboxy-deprotections under basic conditions are known by those of ordinary skill in the art. For example, the compound of formula (11) may be treated with a suitable base, such as lithium hydroxide (LiOH) for a period of time sufficient to remove the carboxy protecting group, for example from about 1 to 24 hours.
In Scheme B, step 6, the ester of formula (12) is coupled with a Fragment CD carboxylic acid of formula: 
wherein Y, R7, R8, R9, R10, R11 and R50 are as defined above and R82 is a base labile protecting group; to provide the compound of formula (13).
For example, the carboxylic acid of formula (12a) is dissolved in a suitable organic solvent, such as DMF, glyme, dioxane, THF, CH3CN, EtOAc, and halohydrocarbons, with dichloromethane being preferred. This solution is then treated with a coupling reagent. Possible coupling reagents include DCC, EDCI, and similar reagents, such as DMAP which activate carboxylic acids towards esterification with alcohols. This solution may then be optionally treated with a suitable base such as solid sodium bicarbonate and then contacted with an ester of formula (12). The concentration of (12a) after these additions should range from about 0.1 M to about 2.0 M. The reaction takes place in the range of from about xe2x88x9230xc2x0 C. to about 60xc2x0 C. with from about 10xc2x0 C. to about 30xc2x0 C. being preferred. The reaction is run for a period of time ranging from about 0.5 to 12 hours. The final concentration of crude product (13) may be used without further purification. Alternatively, the compound of formula (13) may be isolated and purified according to procedures well known in the art such as extraction, evaporation, chromatography and recrystallization.
In Scheme B, step 7, the compound of formula (13) is oxidized with a suitable oxidizing agent to provide the sulfone or sulfoxide of formula (14).
A suitable oxidizing agent is an agent capable of converting the sulfide of formula (13) into the sulfone of formula (14), while inert to the epoxide moiety of the molecule. Suitable oxidizing agents include potassium peroxomonosulfate (Oxone), m-CPBA, methyltrioxorhenium(VII), and magnesium monoperoxyphthalate, with Oxone being preferred.
For example, the sulfide of formula (13) is treated with a suitable base, such as sodium bicarbonate followed by a suitable oxidizing agent, such as Oxone. The reaction is carried out in a suitable solvent, such as acetone, DMF, glyme, dioxane, CH3CN, alcohols, THF, EtOAc, halohydro-carbons, chlorobenzene, and toluene, with acetone being preferred. Generally, the reaction is carried out at temperatures of from about xe2x88x9230xc2x0 C. to about 50xc2x0 C. with from about xe2x88x9210xc2x0 C. to about 10xc2x0 C. being preferred. Generally, the reaction requires from about 15 minutes to about 5 hours. Crude sulfone or sulfoxide (14) may be used without further purification. Alternatively, the sulfone or sulfoxide of formula (14) may be isolated and purified according to procedures well known in the art such as extraction, evaporation, chromatography and recrystallization.
In Scheme B, step 8, the sulfone or sulfoxide of formula (14) is deprotected with a suitable deprotecting agent to provide the amine of formula (14a).
A suitable deprotecting agent is an agent capable of removing the base labile substituent R82 on the compound of formula (14) while inert to the epoxide moiety of the molecule. Suitable deprotecting agents include bases such as secondary and tertiary amines and inorganic bases, for example, piperidine, morpholine, dicyclohexylamine, p-dimethylaminopyridine, diisopropylethylamine, and the like, with piperidine being preferred. The reaction is carried out in a suitable solvent such as DMF, glyme, dioxane, CH3CN, alcohols, THF, EtOAc, halohydrocarbons, chlorobenzene, or toluene. Generally, the reaction is carried out at a temperature ranging from about 0xc2x0 C. to about 120xc2x0 C. Generally, the reaction requires from about 1 to 72 hours. The compound of formula (IIB) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization. Alternatively, the compound of formula (14a) is isolated and may be further cyclized with a cyclizing agent to provide a compound of formula (IIB).
Typically, once the compound of formula (14) is deprotected, it undergoes spontaneous cyclization. However, some particular compounds of formula (14) may require an additional cyclization step. Also, for example, the sulfide of formula (13), although much less active than its oxidized counterpart, upon removal of the base-labile protecting group may be cyclized with a second suitable cyclizing agent, such as 2-hydroxypyridine to form a compound of formula (IIB). For example, the sulfide of formula (13), or alternatively a selected compound of formula (14a), is heated in a suitable solvent, such as DMF at about 60xc2x0 C. for several days in the presence of piperidine and 2-hydroxypyridine. The compound of formula (IIB) is isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
In Scheme B, step 9, the epoxide of formula (IIB) is optionally treated with a halohydrin forming reagent to produce the halohydrin of formula (IIC), where Hal is halogen, preferably chlorine.
A xe2x80x9chalohydrin forming reagentxe2x80x9d is any agent capable of coverting the epoxide moiety of compound (IIB) to the halohydrin moiety of compound (IIC). Suitable halohydrin forming reactions are disclosed in PCT Intnl. Publ. No. WO 96/40184, published Dec. 19, 1996 and PCT Intnl. Publ. No. WO 98/09988, published Mar. 12, 1998. For example, the epoxide of formula (IIB) is treated with a suitable halo-acid, such as hydrochloric acid in a suitable organic solvent or solvent mixture, such as dimethoxy-ethane/water. The mixture is then stirred at a temperature ranging from about 10xc2x0 C. to about 50xc2x0 C. for a period of time ranging from about 6 to 36 hours. The mixture is then neutralized with a suitable base or buffer, such as potassium carbonate. The halohydrin of formula (IIC) is isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
In Scheme B, step 10, the halohydrin of formula (IIC) is reacted with a glycinating agent to provide the glycinate ester of formula (IID).
A xe2x80x9cglycinating agentxe2x80x9d is any agent capable of converting the halohydrin of formula (IIC) into the glycinate ester of formula (IID). Suitable glycinating reactions are disclosed in PCT Intnl. Publ. No. WO 98/08505, published Mar. 5, 1998. For example, the halohydrin of formula (IIC) is coupled with N-(tert-butoxycarbonyl)glycine (Boc-Gly) under coupling conditions well known in the art. For example, the halohydrin of formula (IIC) is contacted with Boc-Gly, dimethylaminopyridine (DMAP) and 1,3-dicyclohexylcarbodiimide (DCC). The resulting mixture is stirred at a temperature ranging from 10xc2x0 C. to 50xc2x0 C. for a period of time ranging from 0.5 to 24 hours. The glycinate ester of formula (IID) is isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
A synthetic scheme for making the Fragment CD carboxylic acids of formula (12a) is set forth in Scheme C. The reagents and starting material are readily available to one of ordinary skill in the art. In Scheme C, all substituents, unless otherwise indicated, are as previously defined. 
In Scheme C, step 1, the Boc-protected amine of formula (15) is deprotected to provide the deprotected amine of formula (16).
For example, the deprotection reaction involves the removal of an amino protecting group by techniques and procedures well known and appreciated by one of ordinary skill in the art. The selection, use, and removal of 10 protecting groups are set forth by Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981). For example, the Boc-protected amine of formula (15) is dissolved in a suitable acid, such as trifluoroacetic acid or hydrochloric acid. Generally, the reaction is carried out at a temperature ranging from about 0xc2x0 C. to about 60xc2x0 C. Generally, the reaction requires from about 1 to 24 hours. The deprotected amine of formula (16) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
The Boc-protected amine of formula (15) is described in Barrow, R. A. et al. J. Am. Chem. Soc. 117, 2479 (1995); PCT Intnl. Publ. No. WO 96/40184, published Dec. 19, 1996; and PCT Intnl. Publ. No. WO 97/07798, published Mar. 6, 1997.
In Scheme C, step 2, the deprotected amine of formula (16) is amino-protected with a base-labile amino protecting group to provide the carboxylic acid of formula (12a).
For example, the protection of an amino group with a base-labile amino protecting group involves the addition of a base-labile amino protecting group by techniques and procedures well known and appreciated by one of ordinary skill in the art. The selection, use, and removal of base-labile amino protecting groups are set forth by Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981). A preferred base-labile amino protecting group is Fmoc. For example, to a solution of the deprotected amine of formula (16) in a suitable solvent, such as dioxane, is added a suitable base, such as sodium bicarbonate, followed by a compound of the formula R82-Cl or R82-ONHS, such as Fmoc-Cl or Fmoc-ONHS succinimide. The mixture may be optionally diluted with a small amount of water and stirred for a period of time ranging from 12 to 48 hours at a temperature ranging from about 0xc2x0 C. to about 60xc2x0 C. The mixture may be quenched with a suitable acid, such as hydrochloric acid. The carboxylic acid of formula (12a) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
For further example, Scheme D illustrates a general synthetic procedure for preparing a cryptophycin compound of formula (II). In Scheme D, all substituents, unless otherwise indicated, are as previously defined. As used herein, the substituent xe2x80x9cHalxe2x80x9d stands for halogen. 
In Scheme D, step 1, a compound of formula (IB) is coupled with a Fragment B amino acid of formula (9g) to provide an alkoxy-protected Fragment AB compound of formula (17) according to the procedure set forth in Scheme B, step 3.
In Scheme D, step 2, an alkoxy-protected Fragment AB compound of formula (17) is alkoxy-deprotected according to the procedure set forth in Scheme B, step 4 to provide a Fragment AB compound of formula (18). Alternatively, the alkoxy-protected Fragment AB compound of formula (17) is deprotected according to techniques and procedures well known to one of ordinary skill in the art. Since the alkoxy-protected Fragment AB compound of formula (17) does not possess an epoxide group as does the corresponding analog in Scheme B, the deprotecting reaction conditions are not required to be as sensitive. For example, an alkoxy-protected Fragment AB compound of formula (17) may be deprotected according to the procedure set forth in Barrow, R. A. et al, J. Am. Chem. Soc. 117, 2479 (1995), which includes 50% aqueous HF in a CH3CN solution.
In Scheme D, step 3, a Fragment AB compound of formula (18) is coupled with a Fragment CD carboxylic acid of the formula: 
wherein R7, R8, R9, R10, R11, R50 and Y are as defined above and Pg is a suitable amino protecting group, according to the procedure set forth in Scheme B, step 6 to provide a Fragment ABCD compound of formula (19). Suitable amino protecting groups are well known by one of ordinary skill in the art and are disclosed in Greene, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, John Wiley and sons, New York (1981), the disclosure of which is hereby incorporated by reference. A particularly preferred amino protecting group is t-Boc.
In Scheme D, step 4, a Fragment ABCD compound of formula (19) is deprotected with a suitable second deprotecting agent to provide the deprotected Fragment ABCD compound of formula (20).
A suitable xe2x80x9csecond deprotecting agentxe2x80x9d is any agent or combination of agents which are effective in removing both the xe2x80x9cPgxe2x80x9d amino protecting group and the xe2x80x9cRp1xe2x80x9d carboxy protecting group, either sequentially or concomitantly. Since the Fragment ABCD compound of formula (19) does not possess an epoxide group as does the sulfoxide or sulfone of formula (14) in Scheme B, step 8, the deprotecting reaction conditions are not required to be as sensitive. For example, a Fragment ABCD compound of formula (19) may be deprotected according to the procedure set forth in Barrow, R. A. et al, J. Am. Chem. Soc. 117, 2479 (1995). The deprotected Fragment ABCD compound of formula (20) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
In Scheme D, step 5, the deprotected ABCD compound of formula (20) is cyclized with a second suitable cyclizing agent according to Barrow, R. A. et al, J. Am. Chem. Soc. 117, 2479 (1995) to form the cyclic alkene of formula (IIA). Alternatively, the deprotected ABCD compound of formula (20) may be cyclized with a suitable cyclizing agent according to Scheme B, step 8. The cyclic alkene of formula (IIA) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
In Scheme D, step 6, the cyclic alkene of formula (IIA) is epoxidized according to the procedures set forth in Scheme B, step 2 or Scheme B1 to provide the epoxide of formula (IIB).
In Scheme D, step 7, the epoxide of formula (IIB) is treated with a halohydrin forming reagent according to Scheme B, step 9 to produce the halohydrin of formula (IIC).
Alternatively, the cyclic alkene of formula (IIA) is contacted sequentially with an epoxidizing agent and a trialkylsilyl chloride according to PCT Intnl. Publ. No. WO 98/09988, published Mar. 12, 1998 to provide the halohydrin of formula (IIC) where xe2x80x9cHalxe2x80x9d is chloro.
In Scheme D, step 8, the halohydrin of formula (IIC) is reacted with a glycinating agent according to Scheme B, step 10, to provide the glycinate ester of formula (IID).
Optionally, on those compounds of formulae (I) or (II) containing basic or acidic functional groups, pharmaceutically acceptable salts of the compounds of formulae (I) or (II) may be formed using standard techniques. For example, the free base may be dissolved in aqueous or aqueous-alcohol solution or other suitable solvent containing the appropriate acid and the salt isolated by evaporating the solution. Alternatively, the free base may be reacted in an organic solvent containing the appropriate acid and the salt isolated by evaporating the solution. Further, the free base may be reacted in an organic solvent in which case the salt separates directly or can be obtained by concentration of the solution or in a solvent such as water which is then removed in vacuo or by freeze-drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.
It should be noted that since one aspect of the invention represents a convergent synthesis to produce a cryptophycin compound of formula (II), alternate sequences of couplings may be utilized. For example, Fragment A may be first coupled to Fragment B to form Fragment AB and Fragment Cxe2x80x2 to Fragment D to form Fragment Cxe2x80x2D. Fragment AB may then be coupled to Fragment Cxe2x80x2D to form Fragment ABCxe2x80x2D.
Preferred embodiments of the processes for preparing compounds of formulae (I) and (II) are given individually below:
(a) G is phenyl, p-fluorophenyl, or p-chlorophenyl;
(b) R1 is chloro and R2 is OH;
(c) R1 is chloro and R2 is glycinate ester;
(d) R1 and R2 are taken together to form an epoxide ring;
(e) R1 and R2 are taken together to form a bond;
(f) R3 is methyl;
(g) R6 is a group of formula (IA) wherein R6a is chloro, R6b is methoxy and R6c is hydrogen;
(h) one of R7 or R8 is hydrogen while the other is methyl;
(i) R7 and R8 are both methyl;
(j) R9 is hydrogen and R10 is C1-C6 methyl;
(k) R11 is hydrogen;
(l) R14 is hydrogen;
(m) R50 is (xe2x95x90O);
(n) Y is O;
(o) the combination of embodiments (a), (b), (f), (g), (h), (j), (k), (l), (m) and (n);
(p) the combination of embodiments (a), (c), (f), (g), (h), (j), (k), (l), (m) and (n);
(q) the combination of embodiments (a), (d), (f), (g), (h), (j), (k), (l), (m) and (n);
(r) the combination of embodiments (a), (e), (f), (g), (h), (j), (k), (l), (m) and (n);
(s) the combination of embodiments (a), (b), (f), (g), (i), (j), (k), (l), (m) and (n);
(t) the combination of embodiments (a), (c), (f), (g), (i), (j), (k), (l), (m) and (n);
(u) the combination of embodiments (a), (d), (f), (g), (i), (j), (k), (l), (m) and (n); and
(v) the combination of embodiments (a), (e), (f), (g), (i), (j), (k), (l), (m) and (n).
To further illustrate the invention the following examples are provided. The scope of the invention is in no way to be construed as limited to or by the following examples. The terms and abbreviations used in the instant examples have their normal meanings unless otherwise designated. For example xe2x80x9cxc2x0 C.xe2x80x9d refers to degrees Celsius; xe2x80x9cNxe2x80x9d refers to normal or normality; xe2x80x9cmmolxe2x80x9d refers to millimole or millimoles; xe2x80x9cgxe2x80x9d refers to gram or grams; xe2x80x9cmlxe2x80x9d or xe2x80x9cmLxe2x80x9d means milliliter or milliliters; xe2x80x9cMxe2x80x9d refers to molar or molarity; xe2x80x9cMSxe2x80x9d refers to mass spectrometry; xe2x80x9cIRxe2x80x9d refers to infrared spectroscopy; and xe2x80x9cNMRxe2x80x9d refers to nuclear magnetic resonance spectroscopy. 
Boc amine (1.69 g, 5.09 mmols) of the formula 
PCT Intnl. Publ. No. WO 97/07798, published Mar. 6, 1997; was dissolved in trifluoroacetic acid (17 ml) and the solution stirred at room temperature under a dry nitrogen atmosphere for 4.75 h and then concentrated in vacuo and dried under high vacuum for 24 h to give the amine salt as a yellow viscous oil (1.76 g, 100%).
[xcex1]D589 xe2x88x9211.54xc2x0 (c 1.04, MeOH); 1H NMR (CDCl3) xcex4 Unit Cxe2x80x2: 7.43 (br s, 3H, NH3+),3.34-3.28 (m, 3-H), 3.18-3.12 (m, 3-Hxe2x80x2), 1.42 (s, 2-Me), 1.36 (s, 2-Me); Unit D: 10.94 (br s, CO2H), 5.23-5.20 (m, 2-H),1.92-1.77 (m, 3H, 3-HHxe2x80x2, 4-H), 1.10 (d, J=5.8 Hz, 5-H3), 0.98 (d, J=5.8 Hz, 4-Me) ppm; IR (CHCl3) xcexd 2963, 1746, 1710, 1678, 1192, 1172 cmxe2x88x921; MS (FAB) 232.2 ([M+1]+, 100). 
To a stirred solution of amine salt of Preparation 1 (5.09 mmols) in dioxane (20 mL) was added sodium bicarbonate (2.14 g,25.5 mmols) followed by FmocCl (1.58 g,6.11 mmols) at room temperature. The mixture was diluted with H2O (4 mL) and stirred for 19 h. The reaction mixture was quenched in 1N aqueous HCl (150 mL) and extracted with EtOAc (2xc3x97100 mL). Combined organics were washed with H2O (100 mL), dried (MgSO4) and concentrated in vacuo to give a yellow gummy solid. The crude product was purified by column chromatography (Biotage-SiO2: gradient elution; 10%-75% EtOAC: Hexanes) to provide Fmoc amine as a pale yellow solid (850 mg, 37%). Product was contaminated with amino acid, which was removed by dissolving the product in EtOAc and stirring with 1N HCl aq for several hours. Organics were dried and concentrated to give product (85:15 product: amino acid).
[xcex1]D589 xe2x88x9215.95xc2x0 (c 0.50, CH2Cl2); 1H NMR (CDCl3) xcex4 Unit Cxe2x80x2: 7.59 (d, J=7.4 Hz, ArH2) , 7.67-7.61 (m, ArH2), 7.43 (t, J=7.3 Hz, ArH2), 7.36-7.30 (m, ArH2), 5.88 (t, J=5.8 Hz, NH), 4.41-4.38 (m, 3xe2x80x2-HHxe2x80x2), 4.35-4.28 (m,4xe2x80x2-H), 3.42 (d, J=6.5 Hz, 3-HHxe2x80x2), 1.27 (s, 2Me), 1.26 (s, 2-Me); Unit D: 8.40 (br s, CO2H), 5.18-5.13 (m, 2-H), 1.87-1.69 (m, 3H, 3-HHxe2x80x2, 4-H), 0.97 (d, J=5,8 Hz, 5-H3), 0.93 (d, J=6.1 Hz, 4-Me) ppm; IR (KBr) xcexd 2959, 2937, 1730, 1540, 1471, 1451, 1307, 1268, 1145, 1128, 759, 741 cmxe2x88x921; UV (EtOH) xcexmax 299 (e=5851), 288 (e=4773), 265 (e=18369), 227 (e=4813) nm; MS (FAB) 454 ([M+1]+, 26); Anal. calcd. for C26H31NO6 requires: C, 68.86; H, 6.89; N, 3.09%. Found: C, 68.92; H, 7.01; N, 3.34%. 
A sample of t-Butyl-3-(R)-benzyl-3-amino-propanoic acid (purchased from oxford Asymmetry, England,  greater than 99% e.e) was dissolved in trifluoroacetic acid (TFA) and then let stirred at room temperature for 4 h. The trifluoroacetic acid was removed in vacuo to give an oily residue which was then triturated with methanol to give a white solid.
TLC: Rfxe2x95x90(CHC13/CH3OH/NH4OH: 6:3.2:0.8)
IR (cmxe2x88x921):
1HNMR(300 MHz, DMSO-d6) d: 7.93 (bs, 2H), 7.32 (m, 5H), 3.63 (t, J=7.2 Hz, 1H), 2.91 (dd, J=5.9 Hz, J=13.6 Hz, 2H), 2.77 (dd, J=8.1 Hz, J=13.6 Hz, 2H)
Anal: Calcd for C12H14NO4: C, 49.15; H, 4.81; N, 4.78. Found: C, 48.87; H, 4.73, N, 4.70. 
A sample of the compound of Preparation 3 was dissolved in 1,4-dioxane/H2O/2.0 NNaOH (2:2:1) at 0xc2x0 C. (ice bath). To this was then added di-t-butyl-dicarboxylate and the ice bath was removed and the resulting reaction mixture was let stirred at room temperature for 18 h. The reaction mixture was then concentrated to about 10 ml and 25 ml of EtOAc was added. To this was then added 0.5 N NaHSO4 to lower the pH of aqueous phase to ca. 2-3. The organic layer was then separated and the aqueous layer was extracted with EtOAc (20 ml xc3x973). The combined EtOAc layer was then washed with water and brine and dried over NaSO4. The solvent was then removed in vacuo to give a pale yellow solid.
TLC: Rf=(CHC13/CH3OH/NH4OH: 6:3.2:0.8)
IR (cmxe2x88x921): 3361, 2985, 1670, 1686, 1526, 1266, 1168, 700.
UV (CH3OH): 258 nm (e=158).
OR: [xcex1]D=xe2x88x92136.71
1HNMR(300 MHz,DMSO-d6) d: 7.20 (m, 5H), 6.75 (d, J=8.6 Hz, 1H), 3.88 (m, 1H), 2.64 (d, J=7.0 Hz, 2H), 2.28 (t, J=5.1 Hz, 2H)1.27 (s, 9H).
Mass(FAB): 280 (M++H).
Anal: Calcd for C15H21NO4: C, 64.50; H,7.58; N,5.01. Found: C, 63.25; H, 7.35, N, 4.99. 
To a solution of allyl (2S)-2-hydroxy-4-methylpentanoate and (3R)-benzyl-3-(tert-butoxycarbonyl)amino-propanoic acid (Preparation 4) in 10 ml of dry methylene chloride at 0xc2x0 C. (ice bath), was added dicyclohexylcarbodiimide and then followed by DMAP. The reaction mixture was then let stirred at room temperature for 3 h (TLC indicated the completion of the reaction). The reaction mixture was then filtered through a small pad of celite and the filtrate was washed with 5% NaHCO3, brine and dried over Na2SO4. The solvent was removed in vacuo and the residue was flash chromatographed on SiO2 (15% EtOAc/hexane) to give the title compound as a clear oil.
TLC: Rf=(20% EtOAc/hexane)
IR (cmxe2x88x921): 2961, 2933, 1742, 1715, 1497, 1366, 1249, 1170, 1127.
UV (CH3OH): 258 nm (e=218).
OR: [a]D=+7.55
1HNMR(300 MHz, CDCl3) d: 7.25 (m, 5H), 5.89 (m, 1H), 5.20-5.36 (m, 3H), 5.10 (dd, J=3.9 Hz, J=9.6 Hz, 1H), 4.65 (d, J=5.4 Hz, 2H), 4.15 (bs, 1H), 2.87 (m, 2H), 2.62 (dd, J=5.6 Hz, J=15.4 Hz, 1H), 2.50 (dd, J=5.0 Hz, J=15.4 Hz, 1H), 1.60-1.85 (m, 3H), 1.40 (s, 9H), 0.95 (d, J=4.3 Hz, 3H), 0.93 (d, J=4.3 Hz, 3H).
Mass(FAB): 434.4 (M++H).
Anal: Calcd for C24H35NO6: C, 66.49; H, 8.14; N, 3.23. Found: C, 66.32; H, 8.29, N, 3.42.